Campylobacter jejuni is a Category B Bioterrorism Agent as classified by the NIH. It is the leading cause of bacterial gastroenteritis in the United States, and is responsible for sporadic disease as well as food-borne and water-borne outbreaks. There are at least 2.4 million cases of C. jejuni gastroenteritis in the U.S. annually, with an incidence exceeding that of Salmonella and Shigella combined (1). C. jejuni infection is also the most common antecedent event to development of Guillain-Barr Syndrome, an acute motor paralysis apparently resulting from an autoimmune response directed at C. jejuni surface antigens. Despite the high prevalence of Campylobacter disease and more than 20 years of study, the mechanisms by which C. jejuni causes disease remain obscure. Several C. jejuni virulence factors have been identified, yet their regulatory mechanisms are largely unknown. Recent evidence implicates DNA methylation as an important signal controlling virulence gene expression in Salmonella and other bacteria. In light of this, our studies on the predicted C. jejuni DNA methylase Cj1461 showed that cj1461 was induced at 37C, the internal temperature of humans, consistent with a role in pathogenic growth adaptation. Further experiments revealed that mutation of cj1461 resulted in the dramatically altered expression of ca. 50-60 proteins, including known virulence factors and additional predicted regulatory proteins such as the orphan response regulator Cj0355. Consequently, Cj1461 appears to be involved in a complex regulatory circuit controlling expression of numerous C. jejuni genes; growth temperature is one of the signals. We now propose further study of gene regulation in C. jejuni, focusing on those proteins that are regulated by the predicted DNA methylase Cj1461 and the orphan response regulator Cj0355. We will use a combination of microarray, proteomics, and biochemical approaches to achieve the goals outlined in these three specific aims: Specific Aim 1. Determine the functional properties of Cj1461 and Cj0355 responsible for modulating virulence gene expression. Specific Aim 2. Identify the downstream targets of Cj1461 and Cj0355 to define their combinatorial impact on gene regulation. Specific Aim 3. Elucidate the upstream factors and signals controlling Cj1461 and Cj0355 expression to reveal the entire regulatory circuit.